The determination of kinetic relationships for reactants and other components in microfluidic systems can be a relatively complex process. In this regard, the inventors and their co-workers have determined a variety of useful methods of determining kinetic information for reactions and other phenomena in such microfluidic systems. For example, xe2x80x9cAPPARATUS and METHODS FOR CORRECTING FOR VARIABLE VELOCITY IN MICROFLUIDIC SYSTEMS,xe2x80x9d W098/56956 by Kopf-Sill et al. provides pioneering methods of obtaining kinetic information for moving reactants, based, e.g., upon the conservation of flux in microscale systems which use electrokinetic forces to move fluids.
The difficulties of determining kinetic information are compounded in high-throughput systems where thousands of test compounds per day can be screened in a single microscale system for activity on one or more selected targets. Pioneering high-throughput screening methods and relevant apparatus are described by the inventors and their co-workers in Knapp et al. xe2x80x9cCLOSED LOOP BIOCHEMICAL ANALYZERSxe2x80x9d (WO 98/45481; PCT/US98/06723); Parce et al. xe2x80x9cHIGH THROUGHPUT SCREENING ASSAY SYSTEMS IN MICROSCALE FLUIDIC DEVICESxe2x80x9d WO 98/00231 and in, e.g., No. 60/128,643, filed Apr. 4, 1999, entitled xe2x80x9cMANIPULATION OF MICROPARTICLES IN MICROFLUIDIC SYSTEMS,xe2x80x9d by Mehta et al.
One of the rate limiting aspects of high-throughput screening in general is the need to sample and re-sample the effect of a given test reagent at a variety of concentrations to provide kinetic or other activity data, such as dose-response information. Irrelevant samples (i.e., those with little or no activity in the system of interest) are often unnecessarily re-sampled to determine whether they provide an activity of interest. The need for multiple sampling strategies effectively increases the number of sampling events that a system performs, decreasing the overall throughput of the system. Furthermore, this resampling can involve multiple xyz spatial translations of a microfluidic sample loader or sample library, or both, to provide access to samples of interest, further reducing system throughput.
Accordingly, it would be extremely useful to be able to acquire kinetic information from one or a few sampling events in a microfluidic system. The present invention provides methods, apparatus and systems for generating and deconvoluting signal information and for reduced resampling in high throughput systems (including both pressure-based and electrokinetic systems), as well as a variety of other features which will become apparent upon complete review of the following.
The present invention provides for the use of signal profile information from one or a few sampling events to produce information regarding kinetics and/or reactant concentrations in a microfluidic system. In particular, the shape of the signal profile can be deconvoluted to provide kinetic information and to provide directed re-sampling of a source of sample materials.
In one set of methods, the dwell time for a system sampler is varied to modulate the profile of a signal profile, thereby providing a basis for activity determination and for limiting the need for re-sampling by the system. The system sampler will often utilize pressure-based sampling elements (e.g., pressure-based pipettor channels), although the methods herein can also be adapted to electrokinetic and other fluid movement systems.
In other methods, the dwell time is not necessarily varied, with kinetic information resulting from the deconvolution of signal profile information. Although not necessary, microfluidic channel geometry can be used to facilitate signal deconvolution, e.g., by first dispersing the sample and then reacting the dispersed sample to produce concentration gradient dependent signal information.
For example, the present invention provides high-throughput methods of sampling fluidic materials in which a plurality of different aliquots of the fluidic material are introduced into a microfluidic cavity (typically a channel, well, chamber, reservoir or other structure of microscale dimensions). These aliquots (which typically include a test material to be assayed for activity on a selected target) are produced by systematically varying dwell time at a source of the fluidic material for a microfluidic sample loader which is fluidly coupled to the microfluidic cavity, or by systematically varying a volume of the material in each aliquot. For example, the percent modulation versus target reagent concentration time can be determined by systematically varying the dwell time for a microfluidic sample loader loading material from a source of a test reagent.
The microfluidic sample loader can be configured in a variety of ways, e.g., as a fluid pressure modulatory channel, an electrokinetic modulatory channel, an electrokinetic controller, a fluid pressure controller (e.g., a vacuum source), or the like. Following loading of a test material into the system by the microfluidic sample loader, the material can be flowed into contact with target compositions in the microfluidic cavity for typical mixing reactions, e.g., with a target material.
The system is particularly useful for examining potential activity modulators (i.e., compounds or compositions which facilitate (activate) or inhibit (repress) a reaction of interest, or between an interaction between two or more moieties which interact to produce a detectable result. Typically, the modulator modulates an activity between one or more reactant and one or more reactant substrate. For example, the target composition or the test composition can include an enzyme or other catalyst, a substrate and/or an activity modulator (it will be appreciated that, e.g., kinetic activity can be determined by varying any one or all of these components, depending on the format of the reaction at issue). For example, the modulator can inhibit (e.g., as a competitive inhibitor) or enhance essentially any reaction conducted in the microfluidic system. In the methods herein, it is typical to measure a signal produced by at least one test composition, a target composition, a reaction modulator, and, an interaction between any of these components. Signals are generally produced by one or more labels in the system, and can be a component of, or released by, a reaction.
Label is typically initially confined in a region xe2x88x92h less than x less than h, as a function of time (t) and spatial position (x) with respect to the peak center (x=0) and the concentration (C) of the label, or of a component corresponding to the label, is equal to xc2xd C0 {erf[(hxe2x88x92x)/(2Dt)xc2xd)]}, where C0 is the initial concentration at time t=0, erf is an error function, and D is a coefficient of overall dispersion. D is equal to the sum of thermal diffusion and Taylor dispersion (DT) in the system. In turn, the Taylor dispersion (DT) is dependent on the dimensions and shape of the microfluidic cavity through which the label is flowed, the flow velocity (u) and the thermal diffusivity (D). Typically, D=K(d2u2)/D, where K is a proportionality factor which is a function of the microfluidic cavity through which the label is flowed and d is a characteristic microfluidic cavity length. For example, where the microfluidic cavity is a circular channel and K=1/192, d is the diameter of the circular channel and D=D+DT.
In the methods herein, a kinetic rate constant is typically determined for a reaction between at least one test reagent and a target reagent. For example, the kinetic rate constant can be determined by establishing a calibration curve relating dwell time to dilution factor using one or more dye with a molecular weight which is similar to the molecular weight of the reaction modulator. For example, where the target composition is an enzyme and the test composition is an enzyme substrate, or a potential enzyme substrate, the method can include contacting the at least one test composition or the target composition with a reaction modulator, and determining a rate constant for a reaction between the enzyme or enzyme substrate in the presence of the reaction modulator. For example, the method can include determining a kinetic rate constant for a reaction between the at least one test reagent and the target reagent after exposure of the at least one test reagent, or the target reagent, or both the at least one test reagent and the target reagent, to a reaction modulator, which modulator modulates the reaction.
In a typical format, the modulator is an inhibitor such as a competitive or uncompetitive inhibitor of a reaction between the enzyme substrate or a potential enzyme substrate and an enzyme. For example, where the kinetic rate constant is for an inhibition kinetic rate constant for a competitive inhibitor (KI), the reaction rate (V) is equal to [E]0[S]kcat/([S]+Km(1+[I]/KI), where [E]0 is an initial enzyme concentration, [S] is the enzyme substrate, or the potential enzyme substrate concentration, kcat is a measure of the enzyme turnover rate, Km is the concentration of the enzyme substrate, or the potential enzyme substrate at which V is xc2xd of a maximum reaction rate and [I] is the concentration of the competitive inhibitor. Relationships where the modulator is an activator, or an xe2x80x9cuncompetitivexe2x80x9d or xe2x80x9cnon-competitivexe2x80x9d inhibitor are also set forth herein.
The library of test reagents (or xe2x80x9cmodulator targetsxe2x80x9d) can be configured in any of a variety of ways. For example, the library can include one or more multiwell plates comprising a plurality of test reagents. Similarly, the library can be present on the microfluidic substrate, e.g., as in a plurality of wells, reservoirs or chambers fluidly coupled to one or more microscale channel in the body of the device. In this format, the one or more wells, reservoirs or chambers collectively or individually include a plurality of test reagents. In another embodiment, the library exists on a solid support that has a plurality of dried or fixed test reagents dried or fixed to the support (in this format, microfluidic systems optionally comprise resolubilization pipettors which are fluidly coupled to other channels in the system). Other formats for library storage such as the use of microfluidic bead arrays and the like can also be used.
Typically, the microscale cavity includes a microfluidic channel. The aliquot of material (which includes a test reagent or control reagents) is typically moved through the microfluidic channel under pressure (although electrokinetic approaches are also useful). In this system, the aliquot includes a test reagent which is dispersed along a length of the aliquot in the channel by thermal diffusion and Taylor dispersion, thereby providing a Gaussian concentration profile for the test reagent. The concentration and distribution of concentration of a test reagent in a series or set of aliquots can be in any of a number of formats. For example, the center of each aliquot can be approximately the same, with the concentration of a test reagent at an edge of each aliquot being varied. Alternately, concentration of a test reagent at a center of each aliquot can be variable. The volume of a plurality of the aliquots can vary systematically along a length of the microfluidic cavity (e.g., along a microfluidic channel).
Similarly, the intensity of a signal (produced, e.g., by a detectable label) associated with some component of the aliquot (or another fluidic material) van be varied along a dimension of the microfluidic cavity as the fluidic material is flowed through the cavity. In one aspect, a plurality of signal profiles from a plurality of fluidic materials are collected to produce a library of signal profiles which can be used to deconvolute kinetic and concentration information from signals produced from known or unknown samples. For example, the library of signal profiles can include a set of look-up tables for the concentration of a modulator, reactant, catalyst, enzyme, or other moiety of interest.
In one class of embodiments, the profile of a signal is used to calculate kinetic or quantitative information by comparing the profile (produced by a reaction, e.g., in the presence of a reaction modulator), to a library of signal profiles. Comparison of the profile of the signal produced by the reaction to the library of signal profiles provides an indication of an effect of, e.g., the modulator, on a kinetic measurement of the reaction or an indication of the amount of a starting material in a reaction. Comparison of the profile of the signal produced by the reaction to the library of signal profiles is typically performed by least square analysis.
Commonly, a fluidic material of interest has an associated signal produced by a detectable label, with the intensity of the signal varying along a dimension of a microfluidic cavity (e.g., a microchannel) as the fluidic material is flowed through the cavity. One way of determining concentration and kinetic data for a reaction modulator from a profile of the signal in the cavity includes iterative estimation of an effect caused by the modulator. For example, an estimated effect of the modulator is compared to a measured effect of the modulator, with the effect of the modulator being re-estimated at the same or a different concentration, with the second estimation of the modulator being compared to the same or a different measured effect of the modulator. Estimations can be iteratively repeated and compared to measured effects of the modulator at the same or additional different concentrations on the reaction. As additional data is acquired, estimations become progressively more accurate and the concentration and kinetic information for the modulator is determined.
The present invention is especially adapted to provide high-throughput methods of determining an activity of a test material in a microfluidic system. In this preferred class of embodiments, a plurality of test materials are sampled from a test material library which includes a plurality of sources of test materials. The sampling includes introducing the plurality of test materials into a microfluidic system with a microfluidic sample loader and mixing the test materials with at least one target material in the microfluidic system. An effect of at least one of the plurality of test materials on the target material is measured and a source of the at least one test material within the plurality of sources of test materials is determined. The source of the at least one test material in the test material library is re-sampled by varying the dwell time of the microfluidic sample loader. Typically, a plurality of aliquots of the test reagent are contacted to the target, with a plurality of signals which result from contacting the test reagent and the target being measured.
Similarly, the invention provides methods of deconvoluting concentration dependent information. In these methods, a first reagent having a dispersed concentration profile is flowed in a first channel. The first reagent is reacted with a second reagent which modulates the activity of the first reagent. A signal produced by the resulting activity of the first reagent is detected, along with a signal profile of the signal. The signal profile is then converted into a concentration-dependent activity of the first reagent.
For example, a library of signal profiles produced by mixing the first or second reagent at known or calculated concentrations with the first, second or a third reagent can be provided. The shape of any unknown signal profile is converted into a concentration-dependent activity of the first reagent by comparison of the signal to the library of signal profiles. Alternately, a value for the effect of the second reagent on the activity of the first reagent can be estimated and the estimated value compared to a measured value for the activity of the second reagent on the first reagent. This process can be reiteratively repeated to provide progressively closer approximations of concentration or kinetic information for any of the components in the system.
In other embodiments, concentration and/or signal profiles, created as described above, are used to provide quantitative information regarding starting reactants. For example, an amount of starting nucleic acid is determined in a PCR reaction by flowing the starting material through a microfluidic channel and measuring the diffusion/dispersion profile of the sample plug, e.g., during the reaction, to provide the amount of starting material.
Integrated systems and devices for practicing the above methods are also provided. For example integrated systems for sampling test reagents and determining concentration-dependent reaction information for the effect of the test reagent on a selected target reagent are provided. The apparatus includes a body having a microfluidic cavity disposed therein. A source of a plurality of test reagents is fluidly coupled to the microscale cavity (and can be internal or external to the body structure), e.g., through a pipettor channel (which can be a pressure-pipettor or an electrokinetic pipettor). A source of at least one target reagent is also fluidly coupled to the microscale cavity. One or more microfluidic conduits (typically microscale channels) are located between the microfluidic cavity, the source of a plurality of test reagents and the source of at least one target reagent. A test reagent sampler is placed in fluidic contact with the one or more microfluidic conduit. One or more computer is operably linked to the test reagent sampler. In a typical configuration, the system includes one or more libraries of potential modulator compounds, a source of an enzyme, a source of a substrate, sources of relevant buffers, and appropriate interconnecting microscale channel networks.
The computer typically includes software relevant to practicing the methods noted above. For example, the software includes a first instruction set controlling the test reagent sampler and directing the test reagent sampler to flow a test reagent from the source of test reagents into the microscale cavity, as well as a second instruction set directing the test reagents into contact with the at least one target reagent in the microscale cavity. A third instruction set controls detection of a signal resulting from contacting the test reagent and the at least one target reagent while a fourth instruction set directs varying dwell time of the test reagent sampler in contact with the source of test reagent and/or varying the volume of a test reagent sample loaded by the test reagent sampler. A fifth instruction set directs resampling of the source of test reagents by the test reagent sampler (optionally, the fifth instruction set is executed by the integrated system concurrent with the fourth instruction set).
A detector monitors the signal produced by contacting the test reagents and the at east one target reagent during operation of the system. Typically, the detector is operably linked to the at least one computer, which also includes signal deconvolution software for converting a detected signal profile into concentration-dependent test reagent-target reagent reaction information, e.g., by calculating any of the relationships for concentration or kinetic information noted above. Similarly, the signal deconvolution software optionally includes an instruction set for comparing a detected signal profile resulting from contacting the test reagent and the at least one target reagent to a library of signal profiles produced by mixing the test or target reagent at known or calculated concentrations with the test, target or an additional reagent. As noted above, the shape of the signal profile is converted into a concentration-dependent activity of the first reagent by comparison of the detected signal profile to the library of signal profiles. The signal deconvolution software optionally includes an instruction set for comparing and estimating a value for the effect of the test reagent on the activity of the target reagent and for comparing the estimated value to a measured value for the activity of the test reagent on the target reagent. The signal deconvolution software optionally includes an instruction set for reiteratively estimating values for the effect of the test reagent on the activity of the target reagent at different concentrations or introduced fluid volumes of the test or target reagent and for comparing the estimated values to measured values for the activity of the test reagent on the target reagent at the different concentrations or introduced volumes. In this embodiment, after each measurement of activity at each concentration or introduced volume of test and target reagent, the measured information is used to refine an estimated value of an effect by the test reagent on the activity of the target reagent at an additional concentration.